CN1325630C - Culture of nematoda without bacteria - Google Patents
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- CN1325630C CN1325630C CN 200410065471 CN200410065471A CN1325630C CN 1325630 C CN1325630 C CN 1325630C CN 200410065471 CN200410065471 CN 200410065471 CN 200410065471 A CN200410065471 A CN 200410065471A CN 1325630 C CN1325630 C CN 1325630C
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Abstract
The present invention relates to a method for cultivating abacterial pine wood nematode, which comprises the procedures: 1: pseudomonas fluorescens GcM5-1A CCTCC No: M 204065 are cultivated and are treated in an inactivation mode in order to obtain dead cells; 2: black pine calluses are cultivated; 3: the black pine calluses are treated in an inactivation mode to obtain dead black pine calluses; 4: eggs of the abacterial pine wood nematode are prepared in order to be standby; 5: different culture solutions are prepared from dead / active calluses, dead bacteria and an amount of abacterial water according to the proportion under the abacterial condition; 6: the prepared eggs of the abacterial pine wood nematode are added in any kind of the obtained culture solutions; then, the culture solutions are placed at the temperature of 15 DEG C. to 40 DEG C. in order to be cultivated for 4 to 20 days in the dark; thereby, the needed pine wood nematode is obtained. The pine wood nematode cultivated by the present invention has higher output and quality than the prior art, and the present invention is convenient for large-scale commercial processes simultaneously.
Description
(1) technical field:
The present invention relates to a kind of cultural method of axenic pine wood nematodes was studied.
(2) background technology:
Pine nematode (Bursaphelenchus xylophilus (Steiner﹠amp; Buhrer) Nickle) be a kind of destructive disease on the pine tree, mainly propagate the black pine of to cause harm (Pinus thunbergii), Japanese red pine (P.densiflora), slash pine (P elliottii), Pinus massoniana Lamb nearly 40 kinds of pine trees such as (P.massoniana) by Monochamus alternatus (Monochamus alternatusHope).Pine nematode makes the mass mortality of pine tree plant, popular causing disaster, and it has caused serious threat for the production of forestry of China.
Though as far back as 20 beginnings of the century just relevant for the record of pine nematode, just begin this disease is carried out comparatively deep research up to entering the seventies, and obtained some achievements.Yet, pine wood nematode is caused the explanation that most of people accepted of still doing nothing so far of the withered mechanism of pine tree, especially the effect of bacterium in pine nematode that pine wood nematode is carried especially view differ.In China, professor Zhao Boguang is to the systematic research of contrasting of the pathogenesis of pine nematode, proposing pine nematode is to infect the compound infectious disease that causes jointly by pine wood nematode and its malignant bacteria of carrying, and determined malignant bacteria be the pine wilt nematode disease mainly cause the factor of withering.At present, the research of pine wood nematode being carried bacteriogenic toxin all has more report at home and abroad, but really then less relatively about the research of the separation and purification of toxin.
Though Plant nematode and insect belong to animal together, it is infecting, more is being bordering on other phytopathy originals such as fungi, bacterium on harm and the research method, so traditionally the research of plant nematode diseases is contained within the plant pathology scope.
According to the classical rule-Koch's Postulates of plant pathology, in the time of studying the Plant diseases that is caused by pathogen, the pure culture that obtains pathogen is the key of whole research.Therefore, study, at first will obtain a large amount of nematodes plant nematode diseases.Obtain a large amount of enough pine wood nematode pure cultures, no matter be to the biological chemistry of pine wood nematode own, physiology or to the malignant bacteria that pine wood nematode is carried with it concern with and the research that causes the toxin that withers of the malignant bacteria of carrying, be vital.
The a large amount of cultured method of nematode mainly contain three kinds, promptly single biological culture, many biological culture and pure culture.
At present mostly the method that pine nematode is carried out artificial culture is that single different live body cultivates, it is single biological culture, its method mainly contains two kinds, a kind of is to be that foodstuff is cultivated (Mamiya with the fungi, 1975 Behavior ofBursaphelenchus lignicolus in the wood of pine seedlings andpathological responses of pine to nematode infection.Trans.86th.Mty.Jpn.For.Soc., 285-286; Clear former friend also, 1970 マ Star material nematodes are invaded と breeding .81 and return Ri Lin Talk, 255-256), another kind then utilizes plant tissue culture technique to cultivate the pine tree callus, is that foodstuff is cultivated (Tamura H.Mamiya is Reproductipn ofBursaphelenchus lignicolus on pine callus.Nematologica.25:149-151 Y.1979) with the pine tree callus then.
The pine wood nematode that causes pine nematode belongs to the sliding sword that looses and belongs to nematode (Bursaphelenchus), can be food with multiple fungi, is easy to cultivate on multiple fungi culture medium breeding (Dozono et al., 1974; Kobayashi et al., 1974,1975; Kondo et al., 1978).Therefore, be the different live body culture method of list of foodstuff with the fungi, be to cultivate the topmost method of pine wood nematode.Since clear former friend also (clear former friend is also. (1970) マ Star material nematode is invaded と breeding .81 and returns a day woods Talk, 255-256) begin to cultivate pine wood nematode and be used for inoculation experiments with filamentous fungus since, research all adopts the Botrytis cinerea (Botrytis cinerea) that grows on the PDA plating medium to cultivate breeding for the foodstuff fungi with pine wood nematode basically.Under 25 ℃ of temperature, about 4-5 days pine wood nematode can finish a generation (Mamiya Y. (1975) Behavior ofBursaphelenchus lignicolus in the wood of pine seedlings andpathological responses of pine to nematode infection.Trans.86th.Mty.Jpn.For.Soc., 285-286).The research of cultivating about the single different live body of pine wood nematode mostly based on this, carry out the improvement of method: Cao Yue, Shen Baikui (Cao Yue, Shen Baikui. the toxicity research of pine wood nematode extract under (1996) artificial culture condition. Nanjing Forestry University's journal, 20 (4): be the foodstuff fungi with Botrytis cinerea (Botrytis cinerea) and loose top drying germ (Sphaeropsis sapinea) respectively 13-16), with the PDA-fungi, corn grain-fungi is a substratum, cultivate pine wood nematode, the result shows no matter the foodstuff fungi is Botrytis cinerea (Botrytis cinerea) or loose top drying germ (Sphaeropsissapinea), and corn grain-fungi culture medium is all high than the nematode production on the planar PDA-fungi culture medium of routine.Yasuharu Mamiya (Yasuharu Mamiya. (1990) Effects of FattyAcids Added to Media on the Population Growth of Bursaphelenchusxylophilus (Nematoda:Aphelenchoididae) .Appl.Ent.Zool., 25 (2): 299-309) oleic acid, linolic acid, palmitinic acid and stearic acid are added the fungi culture medium that is used for cultivating pine wood nematode, find that oleic acid can improve the egg laying amount of pine wood nematode.Ceng Yongsan (Ceng Yongsan. Feng Zhixin. the Study on artificial culture of (1996) pine wood nematode.Short happy agrotechnique institute journal, 9 (1): 44-49) grade is cultivated nematode with pure pine sawdust or pine sawdust-PDA, and pine sawdust carried out three kinds of different processing, promptly without sterilization, surface sterilization and sterilization fully, found that nematode nematode on aseptic substratum is irreproducible fully, having on the bacterium culture medium and can breed, and the effect of breeding nematode becomes positive correlation with the quantity of fungi; With different foodstuff fungus culture nematode the time, the culture effect of finding Ceratocystis fimbriata Strains (Ceratocystis sp), chaetomium (Chaetomium sp) and sickle-like bacteria (Fusarium sp) will obviously be better than Botrytis cinerea (Botrytis cinerea) and the pestalotia bacteria (Pestalotia sp) used always.
At present, also few about the report that is as good as cultivation of pine wood nematode.Abroad, by Bolla and work together and proposed a kind of method (Bolla RI that cultivates axenic pine wood nematodes was studied that utilizes in nineteen eighty-two, JordanW. (1982b) Cultivation of the pine wilt nematode, Bursaphelenchusxylophilus, in axenic culture media.J.Nematol., 14 (3): 377-381), this method is with SP/YE supplemental medium (SP/YE supplemented medium) and improved Caenorhabditis substratum (modified Caenorhabditi medium) pine wood nematode to be cultivated.Discoveries such as Bolla, one times of required time of line insect population quantity growth, the former is the latter's 1/2; The maximum population quantity that nematode can reach, the former than the big 1.5-2 of the latter doubly.Based on this result of study, Cao Yue (Cao Yue, Shen Baikui. the toxicity research of pine wood nematode extract under (1996) artificial culture condition. Nanjing Forestry University's journal, 20 (4): 13-16) when pine wood nematode being as good as cultivation, adopt the SP/YE supplemental medium, also obtained culture effect preferably.Yet be that Bolla (1982b) or the research of Cao Yue (1996) all show, line insect population in cultivation, occur and reach the phenomenon that begins to descend after the maximum value.This is with before and after susceptible naturally pine death, phenomenon unanimity (the Kondo E that similar pine wood nematode population scale begins to reduce also appears, Ishibashi N. (1978) Ultractructural differences betweenthe propagative and dispersal forms in pine wood nematode, Bursaphelenchus lignicolus, with reference to the survival.Appl.Entomol.Zool., 13:1-11).Bolla (seing before in 1982 sources) etc. regulate the SP/YE supplemental medium of cultivating nematode with 100mM NaOH every day, also fail to prolong the stage of nematode population growth.Above-mentioned cultural method, Yi Qian method relatively, obtained good result, but all have following weak point: 1, prescription material therefor price is more expensive, and be difficult for assorting, individual size of the axenic pine wood nematodes was studied of 2, turning out and the wilder pine wood nematode of breeding potential are much lower with corresponding fungi or black pine callus, and 3, occur line insect population in cultivating and reach after the maximum value beginning phenomenon of decline rapidly.Still do not understand at present the reason of this phenomenon, therefore still do not have solution.
Existing immediate technology is Guo Daosen (Guo Daosen, Cong Peijiang, Li Li, Zhao Boguang. (2002) pine wood nematode is carried the mensuration of number of bacteria and the cultivation of axenic pine wood nematodes was studied. University Of Qingdao's journal, 15 (4): 29-31) wait the axenic pine wood nematodes was studied method of reporting of on the black pine callus, cultivating, this method is to utilize plant tissue culture technique to cultivate the pine tree callus, cultivates nematode with callus then.This method is more effective than method commonly used, has both overcome foodstuff fungi method complex operation, has easily polluted, is difficult to get rid of foodstuff fungi or sterilizing agent to shortcomings such as experimental result may exert an influence, and it is short to be as good as the used culture cycle of live body culture method again, and it is high that output is wanted.Cultivate axenic pine wood nematodes was studied at present at home and adopt this method more.
The concrete steps of this method are:
1. cultivate the black pine callus
Choose the black pine mature seed, rinse well, peel off kind of a skin, in 75% ethanolic soln, soak 40s, again at 0.1%HgCI with tap water
2Handle 4min in the solution, aseptic water washing 5 times divests endosperm, takes out mature embryo, places on the callus inducing medium in 25 ℃ of following dark culturing.Treat to move to after callus grows succeeding transfer culture on the 1P2MS solid medium that contains an amount of hormone (Gao Rong, Zhao Boguang. prevent the method [J] of black pine ectoplast and callus brown stain thereof. Nanjing Forestry University's journal, 2001,25 (5): 75-77.).
2. aseptically process of pine wood nematode and cultivation
Select the stronger Nt of virulence and two worm strains of Nm as for the strain of examination worm through inoculation test, wherein Nt is from black pine, and Nm is from Pinus massoniana Lamb.The pine wood nematode of cultivation on the Botrytis cinerea bacterium adopts the graceful funnel method of shellfish to separate.The pine wood nematode that obtains is used 3%H earlier
2O
2Handle 10min, respectively handle 2h with 0.5% Vetstrep and 0.5% gentamicin sulphate again.All change clothes 3 times after each the processing with sterilized water.Under stereoscopic microscope,, move on the black pine callus through 20～30 of the nematodes of above-mentioned processing with the fine needle picking of the bacterium of going out, put 25 ℃ of following dark culturing.The nematode turned out earlier with the beef-protein medium check, is strictly and uses black pine callus succeeding transfer culture again after aseptic.During test, go out axenic pine wood nematodes was studied, make the nematode suspension of 16000/mL with aseptic water washing.With the microsyringe line taking worm suspension 25 μ L (containing 400 of nematodes) of the bacterium of going out, dropping in the fresh weight that is incubated in the Erlenmeyer flask is the callus surface of 3g, and 25 ℃ of following dark culturing are put in 30 bottles of each worm strain inoculations.
The weak point of this method is: 1, growths such as volume, quantity and nematode egg laying amount, the breeding index aspect from cultivating the pine wood nematode that obtains, still having than the pine wood nematode in the susceptible naturally pine tree can not satisfactory part.2, this method still is not easy to large-scale industrial production.
(3) summary of the invention:
The present invention is intended to overcome the deficiencies in the prior art part, provide a kind of energy a large amount of, fast, the convenient and cultural method of the axenic pine wood nematodes was studied that cost is low.
Technical solution of the present invention is as follows:
Separate the bacterium that pine wood nematode is carried under natural condition and choose Pseudomonas fluorescens (Pseudomonas fluorescens) GcM5-1A bacterial strain.
The biological characteristics of this bacterial isolates:
(1) cultivate proterties: colonial morphology: this bacterial strain all can be grown in 10～41 ℃ NB nutrient solution, and the bacterium colony circle is faint yellow, translucent, chromogenesis not, surface wettability, smooth, neat in edge.Be grown to 25～35 ℃; In the NB of pH4～4.5 nutrient solution, do not grow, in pH5～9 scopes, all can grow optimal pH 7～8.Individual morphology: cell is shaft-like, in some cell cavity is arranged, size 0.5～0.6 * 1.0～2.0 μ m.The Gram-negative reaction, motion has a polar flagella.No pod membrane.
(2) physiological and biochemical property: the Gram-reaction feminine gender, strict aerobic growth is the oxidation acid-producing on Xiu-Li Fusen oxidation-fermentation medium.Chromogenesis not.The poly-particle is arranged in the cell.Catalase and oxydase are all positive.Do not produce arginine dihydrolase.Gelatin hydrolysate does not utilize the nitrate anaerobic growth, the Phospholipid hydrolase of not laying eggs.Hydrolyzed starch not.Glucose, wood sugar, ribose, inositol and Beta-alanine growth be can utilize, but trehalose and tartrate growth do not utilized.
This bacterial strain is deposited in Chinese typical culture collection center on September 5th, 2004, and it abbreviates CCTCC as, and deposit number is CCTCC No:M 204065.
1, above-mentioned bacterial strains is cultivated acquisition bacteria in viable somatic cells.
Obtain dead cell thereby 2, above-mentioned acquisition bacteria in viable somatic cells is carried out inactivation treatment, standby.
3, separate, cultivate pine wood nematode and prepare the axenic pine wood nematodes was studied ovum, standby.
4, cultivate the black pine callus, standby.
Obtain dead black pine callus thereby 5, above-mentioned acquisition black pine callus is carried out inactivation treatment, standby.
6, under the aseptic condition, each lay-by material that obtains with step 2 ~ 5 is mixed with different nutrient solutions in following ratio with an amount of sterilized water:
The dead callus of aseptic exsiccant: dead bacterium (mass ratio) is 5 ~ 100: 1
Callus alive: dead bacterium (mass ratio) is 100 ~ 2000: 1
7, in arbitrary nutrient solution that step 6 obtains, add the axenic pine wood nematodes was studied ovum for preparing in the step 3, place temperature: 15-40 ℃, secretly cultivate 4-20d after, required axenic pine wood nematodes was studied.
Below above steps is further described:
The cultivation of bacterium thalline in the step 1:
Can adopt general NA substratum to carry out flat board cultivates or slant culture.Cultural method is cultural method commonly used.As: take by weighing NA nutrient agar medium powder 32g and be dissolved in the 1000mL distilled water, heated and boiled on electric furnace is sub-packed in the test tube then, in 121 ℃, 1.05kg/cm
2Sterilization 20min cultivates with dull and stereotyped.Can cultivate with the NB liquid nutrient medium during industrial production.The nutrient solution of cultivating after the back will be cultivated with bacterium filter membrane (the about 0.5 μ m of film hole diameter) filters.Get the bacterium thalline.
In the above-mentioned culturing process, the pH value of substratum is 7.0 ± 0.2,28 ± 0.1 ℃ of temperature, slant culture time are 48h, and the liquid culture time is 4 days.
The acquisition of Pseudomonas fluorescens dead cell in the step 2: can adopt chemical process or lyophilize, physics methods such as uviolizing obtain.By the disinfectant in the chemical process bacterium thalline of step 1 gained is handled, or with the bacterium thalline (bacterium liquid) of step 1 gained under aseptic condition, by cryogenic freezing, drying, the dead bacterium of aseptic exsiccant, or with the bacterium thalline (bacterium liquid) of step 1 gained thus place ultraviolet ray down irradiation obtain dead bacterium.As: adopt the trichloromethane method in the chemical process, specific practice is as follows:
A. the somatic cells that cultivation is obtained places test tube or flask, soak an amount of trichloromethane (soak into but do not drip) with the absorbent cotton of bacterium of going out and fill in test tube or flask mouth, after tampon beyond the Great Wall; Place about 2-4h in the sterilisable chamber;
B.2h after, be soaked with trichloromethane absorbent cotton with the aseptic nipper taking-up, test tube mouth or flask mouth are opened wide, in sterilisable chamber, place 2-4h, after treating the trichloromethane volatilization to the greatest extent in the test tube, get 1 ring lawn with transfering loop, insert on the NA slant medium that has prepared, place 28 ℃ constant incubator to cultivate 48h;
C.48h the back observes on the above-mentioned NA slant medium whether bacterial growth is arranged, if do not have, shows that then bacterium is killed by trichloromethane, and dead bacterium can be standby; Otherwise sterilization again.
Adopting the trichloromethane method in the above-mentioned chemical process is one of best approach of being convenient to suitability for industrialized production.This method is simple, effective.
The preparation of axenic pine wood nematodes was studied ovum in the step 3: can adopt known method to cultivate pine wood nematode, treat that it lays eggs, adopt sterilant such as mercuric chloride, antibiotic to separate and obtain aseptic ovum.Adopt H
2O
2It is a preferable methods.Experiment showed, H
2O
2With respect to mercuric chloride, antibiotic etc. not only sterilization effect good and since ovum and nematode to H
2O
2The resistance difference bigger, so use the H of high density
2O
2Kill bacteria and do not influence the hatching rate of ovum fully.Its specific practice is as follows:
A. separate pine wood nematode: will go up the pine wood nematode of cultivating Botrytis cinerea (Botryis cinerea) and separate with the graceful funnel method of shellfish.Centrifugal the pine wood nematode that is separated to, identify, standby;
B. with above-mentioned centrifugal, the pine wood nematode after the evaluation is added on the 2% good dull and stereotyped nutrient agar of prepared beforehand, seals culture dish with sealing film, inserts in 27 ℃ the constant incubator and cultivates 6-24h;
C. in the training period, observe the situation of laying eggs of pine wood nematode; When the nematode egg laying amount reaches the experiment necessary requirement, under aseptic condition, inhale an amount of sterilized water with the suction pipe of the bacterium of going out pine wood nematode on the nutrient agar and ovum are wherein all washed in the centrifuge tube of the bacterium of going out, splash into 30% H
2O
2(with the volume ratio of ovum suspension be 1: 1), under 15 ℃, leave standstill 10min, examine under a microscope the death condition of nematode;
D. treat that nematode is most of dead, with aseptic water washing 3-5 time, add sterilized water at every turn after, be placed on centrifugal 3-4min on the whizzer after shaking up, wait to wash down in the centrifuge tube behind the hydrogen peroxide, filter with 200 aseptic order stainless steel filter sieve, repeat to filter 4-6 time, till most of nematode is filtered out;
E. check that with the NA slant medium for preparing in advance line eggs has or not bacterium; If bacterium, then abandon this experiment and carry out again.
The cultivation of black pine callus in the step 4: can adopt known cultural method to cultivate the black pine callus.As following method:
A. culture medium preparation
Adopting the 1/2MS substratum is minimum medium, additional KT 4.0mg/L, BA 4.0mg/L, sucrose 30g/L is 5.86 with the HCl of 1mol/L and the NaOH adjusting pH value of 1mol/L, adds agar powder 6.5g/L again, be sub-packed in after the heating for dissolving in the triangular flask of 100mL, in 121 ℃, 1.05kg/cm
2Sterilization 15min obtains the substratum of black pine callus induction.
B. adopting the 1/2MS substratum is minimum medium, additional NAA 0.5mg/L, IBA 0.1mg/L, GA3 1.0mg/L, 2,4-D 1.0mg/L, LH 100mg/L, sucrose 30g/L, with the HCl of 1mol/L and the adjusting pH value of 1mol/LNaOH is 5.86, adds agar powder 6.5g/L again, is sub-packed in after the heating for dissolving in the triangular flask of 100mL, in 121 ℃, 1.05kg/cm
2Sterilization 15min obtains the substratum that black pine callus succeeding transfer culture is used.If do not add agar powder after regulating the pH value, obtain the liquid nutrient medium that the unicellular shaking culture of black pine is used after the packing sterilization.
C. the black pine callus induces
The black pine seed is contained in respectively in the small beaker of 50mL, adds a small amount of liquid detergent, seal tight rim of a cup, under flowing water, rinse well with gauze.Shell the back with aseptic water washing 3 times, 75% ethanolic soln surface sterilization 3 times, each 40s uses aseptic water washing 3 times again, afterwards with 0.1% mercuric chloride solution processing 4min, aseptic water washing 5 times.On Bechtop, use the tweezers of the bacterium of going out in the culture dish of bacterium that went out, to divest endosperm, take out mature embryo, it is inserted callus inducing medium, every bottle graft 4-6, place dark culturing in 27 ℃ of incubators.Through about 10d, with well-grown, individuality promptly not contaminated and that brown stain do not take place is transferred to and is kept in the subculture medium and breed.
D. the succeeding transfer culture of black pine callus
In the aseptic processing room, callus is cut into the tissue block of soya bean size, insert on the subculture medium, insert the 3-4 piece in each triangular flask, place 27 ± 2 ℃ of thermostat containers secretly to cultivate, once every the 20-30d succeeding transfer culture.
The acquisition of dead black pine callus in the step 5: can adopt chemical process or lyophilize, physics methods such as uviolizing obtain.By inactivator in the chemical process such as monobromethane the black pine callus that obtains in the step 4 is handled, also can be under aseptic condition with the black pine callus of step 4 gained, by cryogenic freezing, drying, the dead black pine callus of aseptic exsiccant, thereby or be placed on ultraviolet ray down irradiation obtain.Cryogenic freezing, desiccating method are one of best approaches of being convenient to suitability for industrialized production.This method black pine callus simple, that obtained is easy to use, preserve, transportation.Specific practice is as follows:
Above step is obtained fresh black pine callus, under aseptic condition, place-40 ℃ of following 12h frozen dryings, take out under the rearmounted room temperature and thaw, carry out lyophilize, get aseptic exsiccant callus.
The preparation of nutrient solution in the step 6: under the aseptic condition, be mixed in proportion and join with each lay-by material that obtains and be mixed with different nutrient solutions in an amount of sterilized water.
In this step, preferred configuration proportion is:
Dead callus: dead bacterium (mass ratio) is 20 ~ 75: 1
Callus alive: dead bacterium (mass ratio) is 400 ~ 1500: 1
Meet the nutritional need of pine wood nematode with the formulated nutrient solution of this ratio, thereby the growing better of pine wood nematode.
In this step, best configuration proportion is:
Dead callus: dead bacterium (mass ratio) is 40 ~ 60: 1
Callus alive: dead bacterium (mass ratio) is 800 ~ 1200: 1
More meet the nutritional need of pine wood nematode with the formulated nutrient solution of this ratio, thus pine wood nematode grow best.
The cultivation of pine wood nematode in the step 7: in above-mentioned arbitrary nutrient solution, add the axenic pine wood nematodes was studied ovum prepared, place under 15-40 ℃, secretly cultivate 4-20d after, required healthy axenic pine wood nematodes was studied.In this step, preferable culture condition is: temperature is 23-31 ℃, dark cultivation, incubation time 4-10d.This culture condition is suitable for growing of pine wood nematode.Thereby per generation pine wood nematode the cultivation required time also shorter.In this step, optimum culturing temperature is 25 ℃-28 ℃, dark cultivation, incubation time 4-5d.This culture condition is most appropriate to growing of pine wood nematode.Thereby per generation pine wood nematode the cultivation required time also the shortest.
It is more effective than existing method that the present invention adopts technique scheme to cultivate axenic pine wood nematodes was studied, and what individual and wild pine wood nematode was cultivated under the situation of carrying disease germs is individual big or small identical or big slightly, and its breeding potential is also higher, thereby output and quality are all higher than prior art.
The method that employing is cultivated pine wood nematode by dead callus and dead bacterium, sterilized water preparation nutrient solution is the best approach that is suitable for large-scale industrial production, studies show that this method is with respect to obviously promoting growing of pine wood nematode with other nutrient solution in the step 6.Simultaneously, this method no matter from the preservation of dead callus, dead bacterium still from their use, transportation, all help large-scale industrial production.
Superiority of the present invention also has sufficient embodiment on accessibility, well-known, and no matter the cultivation of callus, bacterium is still preserved and not only needed suitable condition but also need suitable technology and time.Turn out axenic pine wood nematodes was studied, all must do these a large amount of previous works, very complicated.The present invention by industrially produce dead callus of aseptic exsiccant and dead bacterium, preserve very conveniently, need not at all to consider whether callus problems such as brown stain take place.Use also very conveniently, need to cultivate pine wood nematode at any time, prepare nutrient solution at any time in proportion, cultivate at any time.
Below manifest the advantage of the present invention on culture effect by following contrast experiment:
The contrast experiment 1: Pseudomonas fluorescens GcM5-1A is to the influence of pine wood nematode egg laying amount
(1) prepare the preparation of axenic pine wood nematodes was studied ovum, dead Pseudomonas fluorescens, preparation NA slant medium, Pseudomonas fluorescens actication of culture, dead Pseudomonas fluorescens successively with aforesaid method, and press following method acquisition bacterial suspension:
A. under the aseptic condition, the Pseudomonas fluorescens GcM5-1A15 ring (internal diameter is about 1mm) with the transfering loop picking is lived is suspended in the 30ml sterilized water, shakes up, and is standby.
B. under the aseptic condition,, be suspended in the 30ml sterilized water, shake up with the dead Pseudomonas fluorescens GcM5-1A15 ring of above-mentioned same transfering loop (after calcination) picking, standby.
(2) prepare 40 aseptic little culture dish (r=1cm, h=1cm), 8 aseptic big culture dish (r=5cm, h=1.5cm), per 5 little culture dish are put into 1 big culture dish, be divided into three groups then, wherein two groups respectively have 3 big culture dish, are labeled as L group (living bacteria) and D group (deadbacteria) with marking pen respectively; Remaining one group has 2 big culture dish, is labeled as C group (control);
(3) in each little culture dish, add 2 axenic pine wood nematodes was studied ovum that prepared with aseptic self-control capillary pipet;
(4) in the little culture dish of L group, respectively add the Pseudomonas fluorescens GcM5-1A suspension alive of 1.5ml then; The dead Pseudomonas fluorescens GcM5-1A suspension that in the little culture dish of D group, respectively adds 1.5ml; C organizes medium and small culture dish and respectively adds the 1.5ml sterilized water;
(5) in sterilisable chamber, finish above-mentioned steps after, with sealing film, put into 27 ℃ thermostat container then and cultivate mouthful the sealing of all big culture dish;
(6) behind the 5d, in sterilisable chamber, observe the situation of little culture dish in each group, observe 5d continuously, write down observed eelworm number of each day, and the eelworm that will add up is chosen with capillary pipet; With the nematode total amount of statistics as the nematode breeding amount in the observation period, the test of significance between handling and contrasting (analyzing) by SPSS11.5.
After observing 5d continuously, the eelworm sum of each group of record is as the breeding amount of nematode in the viewing duration.After the result carried out statistical study, the result was as follows:
Table 1 Pseudomonas fluorescens GcM5-1A is to the influence of pine wood nematode egg laying amount
| Handle | N (nematode logarithm) | Each handles nematode egg laying amount mean+SD (individual) |
| The dead Pseudomonas fluorescens of sterilized water Pseudomonas fluorescens alive | 3 5 5 | 8.67＋2.08a 34.20±5.97b 35.40±81b |
Annotate: the letter in the digital upper right corner is a Duncan ' s multiple comparisons assay, and alphabetical difference represents that difference reaches conspicuous level between the two.(α=0.05) (down together)
As can be seen from Table 1, add the egg laying amount mean value maximum of the pine wood nematode of dead Pseudomonas fluorescens GcM5-1A cultivation, the difference of pine wood nematode egg laying amount is little in each observation group; The egg laying amount mean value of the pine wood nematode that the Pseudomonas fluorescens GcM5-1A that add to live cultivates is slightly littler than the egg laying amount mean value of the pine wood nematode that dead Pseudomonas fluorescens GcM5-1A cultivates, but the differing greatly of pine wood nematode egg laying amount in each observation group; The pine wood nematode egg laying amount mean value of cultivating with sterilized water is all little than the above two.
By table 1 test of significance result as can be known: all there were significant differences with the pine wood nematode egg laying amount of cultivating with sterilized water to use the egg laying amount of the pine wood nematode that dead Pseudomonas fluorescens GcM5-1A and the Pseudomonas fluorescens GcM5-1A that lives after trichloromethane is handled cultivate respectively, illustrates that the both has significant promoter action to the egg laying amount of pine wood nematode; But do not have significant difference between the egg laying amount of the pine wood nematode of cultivating with both respectively, illustrate Pseudomonas fluorescens GcM5-1A whether death the egg laying amount of pine wood nematode is had no significant effect.
The contrast experiment 2: black pine callus and the influence of several different developing mediums of Pseudomonas fluorescens to the pine wood nematode egg laying amount
Be prepared the preparation of axenic pine wood nematodes was studied ovum, dead Pseudomonas fluorescens, preparation NA slant medium, Pseudomonas fluorescens actication of culture, dead Pseudomonas fluorescens, the preparation and the acquisition bacterial suspension of black pine callus successively with preceding method, then,
A. prepare 90 aseptic little culture dish (r=1cm.H=1cm), 18 aseptic big culture dish (r=5cm, h=1.5cm), per 5 little culture dish are put into 1 big culture dish, be divided into seven groups then, wherein three groups respectively have 4 big culture dish, are labeled as DD group (dead calli+dead bateria), LL group (living calli+living bacteria), LC group (living calli), LCDB with marking pen respectively and organize (livingcalli+dead bacteria); Remaining three groups respectively have 2 big culture dish, are labeled as DB group (deadbateria), DC group (dead calli) and C group (control) respectively;
B. in each little culture dish, splash into the black pine callus liquid subculture medium of 1.0ml with aseptic straw;
C. use aseptic self-control capillary pipet (except 15 little culture dish of LC group) in each little culture dish to add 2 axenic pine wood nematodes was studied ovum that prepared;
D. in each little culture dish of LC group, add 2 pine wood nematode ovum with aseptic self-control capillary pipet without aseptically process; After respectively add the 0.01g black pine callus of living again successively, the 1.5ml sterilized water;
E. in the little culture dish of LL group, respectively add 0.01g respectively adds 1.5ml again in little culture dish Pseudomonas fluorescens GcM5-1A suspension alive; In the little culture dish of DD group, respectively add the dead black pine callus of 0.01g, after respectively add the dead Pseudomonas fluorescens GcM5-1A suspension of 1.5ml again; In the little culture dish of LCDB group, respectively add 0.01g without the refrigerated callus of living, after respectively add the dead Pseudomonas fluorescens GcM5-1A suspension of 1.5ml again; In the little culture dish of DC group, respectively add the dead black pine callus of 0.01g, the 1.5ml sterilized water; The dead Pseudomonas fluorescens GcM5-1A suspension that in the DB group, respectively adds 1.5ml; Respectively add the 1.5ml sterilized water in the C group;
F. after in sterilisable chamber, finishing above-mentioned steps, the mouth of all big culture dish is sealed, put into 27 ℃ thermostat container then and cultivate with sealing film;
G.5-7d after, in sterilisable chamber, observe the situation of little culture dish in each group, observe 5d continuously, write down observed eelworm number of each day, and the eelworm that will add up is chosen with capillary pipet; With the nematode total amount of statistics as the nematode breeding amount in the observation period, the test of significance between handling and contrasting (analyzing) by SPSS11.5.
After observing 5d continuously, the eelworm sum of each group of record is as the breeding amount of nematode in the viewing duration.After the result carried out statistical study, the result was as follows:
Several different developing mediums of table 2 black pine callus and Pseudomonas fluorescens are to the influence of pine wood nematode egg laying amount
| Handle | N (nematode logarithm) | Each handles nematode egg laying amount mean+SD (individual) |
| The dead bacterium of the dead bacterium of the dead callus of sterilized water adds the dead bacterium of dead callus and adds callus alive callus (the bacterium ovum is arranged) bacterium that lives that lives and add callus alive | 4 5 4 6 6 7 6 | 9.00＋2.94a 35.00±6.82b 42.00±4.97bc 48.00±13.6c 48.60±5.18bc 49.71±11.38c 54.83±9.20c |
Table 2 as can be seen, the pine wood nematode egg laying amount that adds Pseudomonas fluorescens cultivation alive with the black pine callus of living is maximum, be successively then live the black pine callus culture have bacterium ovum, dead bacterium to add callus alive, dead Pseudomonas fluorescens adds dead black pine callus, dead Pseudomonas fluorescens, dead black pine callus, the egg laying amount that sterilized water is cultivated is minimum.Wherein add the difference maximum of pine wood nematode egg laying amount in each observation group that dead black pine callus, dead bacterium add callus culture alive with dead Pseudomonas fluorescens, the difference minimum of pine wood nematode egg laying amount in each observation group that the contrast sterilized water is cultivated, the pine filaria egg laying amount of other developing medium all has certain difference in each observation group.
Test of significance result by table 2, add Pseudomonas fluorescens alive with the black pine callus of living respectively as can be seen and add dead black pine callus with dead Pseudomonas fluorescens, dead bacterium adds between the egg laying amount of axenic pine wood nematodes was studied of callus culture alive does not have significant difference, same three and between the egg laying amount that the bacterium pine wood nematode is arranged with the black pine callus culture of living does not have difference, but above-mentioned four kinds of developing mediums all and between the egg laying amount of the axenic pine wood nematodes was studied of cultivating with other medium have significant difference, illustrate that these four kinds of developing mediums can promote laying eggs of pine wood nematode significantly.Plant between the developing medium variantly with the egg laying amount of the axenic pine wood nematodes was studied of dead black pine callus culture and first three, but difference is not remarkable; With not having notable difference between the egg laying amount of the axenic pine wood nematodes was studied of dead black pine callus culture and the egg laying amount with the axenic pine wood nematodes was studied of dead microbial culture, but the effect that also has remarkable promotion pine wood nematode to lay eggs with respect to sterilized water.
The contrast experiment 3: the influence that black pine callus and several different developing mediums of Pseudomonas fluorescens grow to pine wood nematode
Be prepared the preparation of axenic pine wood nematodes was studied ovum, dead Pseudomonas fluorescens, preparation NA slant medium, Pseudomonas fluorescens actication of culture, dead Pseudomonas fluorescens, the preparation and the acquisition bacterial suspension of black pine callus successively with preceding method, then,
A. prepare 25 aseptic little culture dish (r=1cm, h=1cm), 5 aseptic big culture dish (r=5cm, h=1.5cm), per 5 little culture dish are put into 1 big culture dish, be divided into five groups then, be labeled as DD group (dead calli+dead bateria), LCDB group (living calli+deadbacteria), LB group (living bacteria), LC group (living calli) DB group (deadbateria) with marking pen respectively;
B. organize the black pine callus liquid subculture medium that splashes into 1.0ml in each little culture dish with aseptic straw to LC;
C. in each little culture dish, add 20 above-mentioned axenic pine wood nematodes was studied ovum that prepared with capillary pipet; After respectively add the 0.01g black pine callus of living again, the 1.5ml sterilized water;
D. in the little culture dish of LC group, respectively add 0.01g black pine callus alive, in the little culture dish of DB group, respectively add the Pseudomonas fluorescens GcM5-1A suspension alive of 1.5ml; In the little culture dish of DD group, respectively add the dead black pine callus of 0.01g, after respectively add the dead Pseudomonas fluorescens GcM5-1A suspension of 1.5ml again; In the little culture dish of LCDB group, respectively add the 0.01g black pine callus of living, after respectively add the dead Pseudomonas fluorescens GcM5-1A suspension of 1.5ml again; The dead Pseudomonas fluorescens GcM5-1A suspension that in the DB group, respectively adds 1.5ml;
E. after in sterilisable chamber, finishing above-mentioned steps, the mouth of all big culture dish is sealed, put into 27 ℃ thermostat container then and cultivate with sealing film;
F.5-7d after, observe the situation of little culture dish in each group in sterilisable chamber, choose each 20 of the female male imagos of the first-generation from each group, the body of measuring female male imago of each generation with micro-ocular micrometer is long, diameter; Put into by the ready next batch culture dish of a-e step with capillary pipet 20 of the eelworms that sucking-off first-generation adult breeds from the little culture dish of each group in addition then, cultivate; Observe each 20 of the female male imagos of the measurement s-generation by above-mentioned same step and method, according to the volume that body is long, the body diameter calculates female male imago of two generations; Formula is 2 ∏ * (d/2) 2 * (l/2)/3, and wherein d is the body diameter of nematode, and l is that the body of nematode is long.With the result who the records analysis that takes statistics, carry out each test of significance (analyzing) between handling by SPSS11.5.
1, the observation measuring result of the female male imago of the first-generation
(1) different developing mediums are to the influence of body length, body diameter and the volume of pine wood nematode female adult worm
The influence that the different developing mediums of table 3 are long to pine wood nematode female adult worm body
| Developing medium | N | The long mean+SD (mm) of body |
| The dead bacterium of the callus dead bacterium of bacterium alive that lives adds the dead bacterium of dead callus and adds callus alive | 20 20 20 20 20 | 0.729800＋0.0558047a 0.855200±0.0273026b 0.858700±0.0218080b 0.880450±0.0195542c 0.890530±0.0206628c |
As can be seen from Table 3, it is maximum adding the long mean value of the female polypide of pine wood nematode that callus alive, dead bacterium add dead callus culture with dead bacterium, is dead bacterium then successively, bacterium lives; And the long individual difference in organizing separately of the female polypide of pine wood nematode that these four kinds of developing mediums are cultivated is all little.With the long mean value minimum of the female polypide of pine wood nematode of the callus culture of living, but bigger in the long individual difference of its group endosome.By the test of significance result as can be known: dead bacterium adds that the long female polypides of cultivating with other three kinds of media of pine wood nematodes of the female polypide of pine wood nematode that dead callus, dead bacterium add callus culture alive are long all a notable difference; Dead bacterium adds dead callus, dead bacterium and adds the female polypide of pine wood nematode that callus two media alive cultivates and do not have notable difference between long; Do not have notable difference between the female polypide of pine wood nematode that bacterium alive and dead bacterium two media are cultivated is long, but both all with the female polypide length of the pine wood nematode of the callus culture of living there is notable difference.Above presentation of results with dead bacterium add dead callus, dead bacterium adds callus culture pine wood nematode alive and more can promote the long growth of the female polypide of pine wood nematode than other three kinds of media.
The different developing mediums of table 4 are to the influence of pine wood nematode female adult worm body diameter
| Developing medium | N | Body diameter mean+SD (mm) |
| The dead bacterium of the callus dead bacterium of bacterium alive that lives adds the dead bacterium of dead callus and adds callus alive | 20 20 20 20 20 | 0.028130＋0.0019445a 0.030860±0.0044518b 0.032670±0.0025695b 0.039510±0.0040150c 0.040320±0.0041623c |
As can be seen from Table 4, add the mean value maximum that callus alive, dead bacterium add the female polypide diameter of pine wood nematode of dead callus culture with dead bacterium, but its individual mesosome diameter difference is bigger; Be dead bacterium then successively, the bacterium that lives, the female polypide diameter individual difference that the female nematode body diameter individual difference that wherein adds microbial culture alive adds dead microbial culture is big; With the female polypide diameter of the pine wood nematode mean value minimum of the callus culture of living, its group endosome diameter individual difference is also minimum.
By table 4 test of significance result as can be known: dead bacterium adds dead callus, dead bacterium adds the female polypide diameter of pine wood nematode of callus culture alive and the female polypide diameter of pine wood nematode of other three kinds of media cultivations all has notable difference; Dead bacterium adds dead callus, dead bacterium adds callus alive notable difference between the two; Do not have living does not have notable difference between the female polypide diameter of pine wood nematode that bacterium and dead bacterium two media cultivate, but both all have notable difference with the female polypide diameter of pine wood nematode with callus culture alive.Above presentation of results with dead bacterium add dead callus, dead bacterium adds callus culture pine wood nematode alive more can promote the female polypide diameter of pine wood nematode than other three kinds of media increase.
The different developing mediums of table 5 are to the influence of female pine wood nematode adult volume
| Developing medium | N | Volume averaging value ± standard deviation (mm 3) |
| The dead bacterium of the callus dead bacterium of bacterium alive that lives adds the dead bacterium of dead callus and adds callus alive | 20 20 20 20 20 | 0.00015322＋0.000096122a 0.00021915±0.000073201b 0.00024207±0.000040577b 0.00036475±0.000075644c 0.00036589±0.000078332c |
As shown in Table 5: adding the long-pending mean value maximum of the female polypide of pine wood nematode that callus alive, dead bacterium add dead callus culture with dead bacterium, is dead bacterium, the bacterium that lives, the callus of living then successively.With regard to body volume difference in organizing, it is bigger with the three kinds of media of bacterium of living that dead bacterium adds dead callus, dead bacterium adds callus alive, and dead bacterium is less with the two kinds of developing mediums of callus of living.From table 5 test of significance result as can be seen: dead bacterium adds dead callus, dead bacterium and adds that the long-pending female polypides of cultivating with other three kinds of media of pine wood nematode of the female polypide of pine wood nematode of callus culture alive are long-pending all a notable difference: do not have notable difference between the female polypide of pine wood nematode that live bacterium and dead bacterium two media are cultivated is long-pending, but both amass with the female polypide of pine wood nematode with callus culture alive all notable difference is arranged.Above presentation of results with dead bacterium add dead callus, dead bacterium adds callus culture pine wood nematode alive and more can promote the increase that the female polypide of pine wood nematode is long-pending than other three kinds of media.
Based on the above results as can be seen: with dead bacterium add that callus alive, dead bacterium add that the body of the female worm of pine wood nematode of dead callus culture is long, the mean value of body diameter and volume all is maximum, and the female polypide of cultivating with other three kinds of media of pine wood nematode is long, body diameter and volume all have notable difference, illustrate with dead bacterium add dead callus, extremely bacterium adds callus culture pine wood nematode alive and more can promote growing of the female worm of pine wood nematode than other three kinds of media.
(2) different developing mediums are to the influence of body length, body diameter and the volume of pine wood nematode male imago
The influence that the different developing mediums of table 6 are long to pine wood nematode male imago body
| Developing medium | N | The long mean+SD (mm) of body |
| The dead bacterium of the callus dead bacterium of bacterium alive that lives adds the dead bacterium of dead callus and adds callus alive | 20 20 20 20 20 | 0.625300＋0.0349060a 0.654700±0.0286321b 0.670450±0.0340239bc 0.685100±0.0218051c 0.689000±0.0221331c |
As can be seen from Table 6: the difference on the pine wood nematode male worm that various different developing mediums are cultivated individual mesosome in each group is long is not very big, wherein adds callus alive, dead bacterium with dead bacterium and adds the body of pine wood nematode male worm of dead callus culture long to organize interior difference be minimum.Add the long mean value maximum of body that dead callus, dead bacterium add the male pine wood nematode of callus culture alive with dead bacterium, the long mean value of pine wood nematode male worm body with dead bacterium, the microbial culture of living is bigger, uses the long mean value minimum of body of the pine wood nematode male worm of the callus culture of living.Test of significance is the result show: adds dead callus, dead bacterium with dead bacterium and adds that the body of pine wood nematode male worm of callus culture alive is long all to have notable difference with the pine wood nematode male worm body of cultivating with the bacterium that lives, the callus two media of living is long, but not obvious with pine line male worm body length difference with dead microbial culture.Do not have notable difference between the pine wood nematode male worm body of cultivating with the bacterium that lives, dead bacterium two media is long, but both with significant difference between the pine wood nematode male worm body length of callus culture alive.Above presentation of results with dead bacterium, dead bacterium add dead callus, dead bacterium adds three kinds of developing mediums of callus alive more can promote pine wood nematode male worm body length with the callus of living, the microbial culture of living growth.
The different developing mediums of table 7 are to the influence of pine wood nematode male imago body diameter
| Developing medium | N | Body diameter mean+SD (mm) |
| The dead bacterium of the callus dead bacterium of bacterium alive that lives adds the dead bacterium of dead callus and adds callus alive | 20 20 20 20 20 | 0.024325＋0.0019404a 0.026795±0.0028153b 0.028970±0.0021428c 0.030735±0.0028077d 0.031864±0.0029002d |
As shown in Table 7: the difference of the first-generation pine wood nematode male worm that various different developing mediums are cultivated individual mesosome diameter in each group is little, and wherein the interior difference of body diameter group with the pine wood nematode male worm of work callus culture is minimum.As can be seen, it is maximum adding the mean value of body diameter that callus alive, dead bacterium add the male pine wood nematode of dead callus culture with dead bacterium, pine wood nematode male worm body diameter mean value with dead bacterium, the microbial culture of living is bigger, uses the long mean value minimum of body of the pine wood nematode male worm of the callus culture of living.The result finds out from test of significance: adding the pine wood nematode male worm body diameter of body diameter and other three kinds of media cultivations that dead callus, dead bacterium add the pine wood nematode male worm of callus culture alive with dead bacterium all has notable difference.Above presentation of results with dead bacterium add dead callus, dead bacterium adds callus culture pine wood nematode alive than other the three kinds broadening that more can promote pine wood nematode male worm body diameter.
The different developing mediums of table 8 are to the influence of pine wood nematode male imago volume
| Developing medium | N | Volume averaging value ± standard deviation (mm 3) |
| The dead bacterium of the callus dead bacterium of bacterium alive that lives adds the dead bacterium of dead callus and adds callus alive | 20 20 20 20 20 | 0.00009823＋0.000022354a 0.00012536±0.000032230b 0.00014912±0.000030663c 0.00017151±0.000037063d 0.00017842±0.000032419d |
Table 8 as can be seen, the first-generation pine wood nematode male worm that various different developing mediums are cultivated in each group between individuality the difference of volume all little.As shown in Table 8: it is maximum adding the mean value of volume that callus alive, dead bacterium add the pine wood nematode male worm of dead callus culture with dead bacterium, is dead bacterium then successively, the bacterium that lives, the callus of living.Test of significance is the result show: adding the pine wood nematode male worm volume of volume and other three kinds of media cultivations that dead callus, dead bacterium add the pine wood nematode male worm of callus culture alive with dead bacterium all has notable difference; Illustrate with dead bacterium and add dead callus culture pine wood nematode more can promote the volume of pine wood nematode male worm than other three kinds of media increase.
Comprehensively long with the upper body, body diameter and volume result be as can be seen: with dead bacterium add that the body that callus alive, dead bacterium add the pine wood nematode male worm of dead callus culture is grown, the mean value of body diameter and volume all is maximum, and the pine wood nematode male worm body of cultivating with other three kinds of media is long, body diameter and volume all have notable difference, illustrate with dead bacterium add dead callus, extremely bacterium adds callus culture pine wood nematode alive and more can promote growing of pine wood nematode male worm than other three kinds of media.
2, the observation measuring result of the female male imago of the s-generation
(1) different developing mediums are to the influence of body length, body diameter and the volume of pine wood nematode female adult worm
The influence that the different developing mediums of table 9 are long to pine wood nematode female adult worm body
| Developing medium | N | The long mean+SD (mm) of body |
| The dead bacterium of the callus dead bacterium of bacterium alive that lives adds the dead bacterium of dead callus and adds callus alive | 20 20 20 20 20 | 0.727800＋0.0390541a 0.859500±0.0253782b 0.866900±0.0208071b 0.887900±0.0149628c 0.888300±0.0168961c |
As seen from the above table: add the long mean value maximum of the female polypide of pine wood nematode that callus alive, dead bacterium add dead callus culture with dead bacterium, be dead bacterium, bacterium alive then successively, and the long individual difference in organizing separately of the female polypide of pine wood nematode that these three kinds of developing mediums are cultivated is all little; With the long mean value minimum of the female polypide of pine wood nematode of the callus culture of living, but bigger in the long individual difference of its group endosome.From the test of significance result of table 9 as can be seen: dead bacterium adds that the long female polypides of cultivating with other three kinds of media of pine wood nematodes of the female polypide of pine wood nematode that dead callus, dead bacterium add callus culture alive are long all a notable difference; Do not have notable difference between the female polypide of pine wood nematode that bacterium alive and dead bacterium two media are cultivated is long, but both all with the female polypide length of the pine wood nematode of the callus culture of living there is notable difference.Above presentation of results with dead bacterium add dead callus, dead bacterium adds callus culture pine wood nematode alive and more can promote the long growth of the female polypide of pine wood nematode than other three kinds of media.
The different developing mediums of table 10 are to the influence of pine wood nematode female adult worm body diameter
| Developing medium | N | Body diameter mean+SD (mm) |
| The dead bacterium of the callus dead bacterium of bacterium alive that lives adds the dead bacterium of dead callus and adds callus alive | 20 20 20 20 20 | 0.028255＋0.0022004a 0.033005±0.0053815b 0.034675±0.0021396b 0.040115±0.00038162c 0.041232±0.00039002c |
As can be seen from Table 10, add the mean value maximum that callus alive, dead bacterium add the female polypide diameter of pine wood nematode of dead callus culture with dead bacterium, but its individual mesosome diameter difference is bigger; Be dead bacterium then successively, the bacterium that lives, the callus of living, the female polypide diameter individual difference that the female nematode body diameter individual difference that wherein adds microbial culture alive adds dead microbial culture is big; The callus group of living endosome diameter individual difference is also minimum.By the test of significance result as can be known: dead bacterium adds dead callus, dead bacterium adds the female polypide diameter of pine wood nematode of callus culture alive and the female polypide diameter of pine wood nematode of other three kinds of media cultivations all has notable difference; Living does not have notable difference between the female polypide diameter of pine wood nematode that bacterium and dead bacterium two media cultivate, but both all have notable difference with the female polypide diameter of pine wood nematode with the work callus culture.Above presentation of results with dead bacterium add dead callus, dead bacterium adds callus culture pine wood nematode alive more can promote the female polypide diameter of pine wood nematode than other three kinds of media growth.
The different developing mediums of table 11 are to the influence of pine wood nematode female adult worm volume
| Developing medium | N | Volume averaging value ± standard deviation (mm 3) |
| The dead bacterium of the callus dead bacterium of bacterium alive that lives adds the dead bacterium of dead callus and adds callus alive | 20 20 20 20 20 | 0.00015407＋0.000031141a 0.00025349±0.000090026b 0.00027461±0.000040206b 0.00037834±0.000071672c 0.00038258±0.000073289c |
As can be seen from Table 11: adding the long-pending mean value maximum of the female polypide of pine wood nematode that callus alive, dead bacterium add dead callus culture with dead bacterium, is dead bacterium, the bacterium that lives, the callus of living then successively.With regard to body volume difference in organizing, it is bigger with the three kinds of media of bacterium of living that dead bacterium adds dead callus, dead bacterium adds callus alive, and dead bacterium is less with the two kinds of developing mediums of callus of living.By the test of significance result as can be known: dead bacterium adds dead callus, dead bacterium and adds that the long-pending female polypides of cultivating with other three kinds of media of pine wood nematode of the female polypide of pine wood nematode of callus culture alive are long-pending all a notable difference; Do not have notable difference between the female polypide of pine wood nematode that bacterium and dead bacterium two media cultivate of living is long-pending, but both amass with the female polypide of pine wood nematode with callus culture alive all notable difference are arranged.Above presentation of results with dead bacterium add callus alive, dead bacterium adds dead callus culture pine wood nematode and more can promote the increase that the female polypide of pine wood nematode is long-pending than other three kinds of media.
The result of comprehensively long with the upper body, body diameter and volume is as can be seen: with dead bacterium add that the body that callus alive, dead bacterium add the female worm of pine wood nematode of dead callus culture is grown, the mean value of body diameter and volume all is maximum, and the female polypide of cultivating with other three kinds of media of pine wood nematode is long, body diameter and volume all have notable difference, illustrate with dead bacterium add dead callus, extremely bacterium adds callus culture pine wood nematode alive and more can promote growing of the female worm of pine wood nematode than other three kinds of media.
The influence that the different developing mediums of table 12 are long to pine wood nematode male imago body
| Developing medium | N | The long mean+SD (mm) of body |
| The dead bacterium of the callus dead bacterium of bacterium alive that lives adds the dead bacterium of dead callus and adds callus alive | 20 20 20 20 20 | 0.631300＋0.0380264a 0.656300±0.0282398b 0.676500±0.0327197c 0.686650±0.0235468c 0.693385±0.0233442c |
As can be seen from Table 12: the difference on the pine wood nematode male worm that various different developing mediums are cultivated individual mesosome in each group is long is not very big, wherein adds dead callus, dead bacterium with dead bacterium and adds the body of pine wood nematode male worm of callus culture alive long to organize interior difference be minimum; It is maximum wherein adding the long mean value of body that dead callus, dead bacterium add the male pine wood nematode of callus culture alive with dead bacterium, is dead bacterium then successively, the bacterium that lives, the callus of living.By the test of significance result as seen: add dead callus, dead bacterium with dead bacterium and add the body of pine wood nematode male worm of callus culture alive and do not have notable difference between long and long, but former three all with between the pine line male worm body length with bacterium alive, callus two media cultivation alive has notable difference with the pine wood nematode male worm body of dead microbial culture.Long and also remarkable with the pine wood nematode male worm body of the microbial culture of living with difference between the pine wood nematode male worm body length of the callus culture of living.Above presentation of results with dead bacterium, dead bacterium add dead callus, dead bacterium adds three kinds of developing mediums of callus alive more can promote pine wood nematode male worm body length with the callus of living, the microbial culture of living growth.
The different developing mediums of table 13 are to the influence of pine wood nematode male imago body diameter
| Developing medium | N | Body diameter mean+SD (mm) |
| The dead bacterium of the callus dead bacterium of bacterium alive that lives adds the dead bacterium of dead callus and adds callus alive | 20 20 20 20 20 | 0.024525＋0.0027493a 0.026895±0.0028153b 0.028975±0.0021584c 0.031010±0.0027493d 0.033221±0.0028474d |
As shown in Table 13: the difference of the s-generation pine wood nematode male worm that various different developing mediums are cultivated individual mesosome diameter in each group is little, wherein uses the interior difference minimum of body diameter group of the pine wood nematode male worm of the callus culture of living.The mean value maximum of body diameter that adds the male pine wood nematode of dead callus culture with dead bacterium is dead bacterium then successively, the bacterium that lives, with the body diameter mean value minimum of the pine wood nematode male worm of the callus culture of living.The result finds out from test of significance: adding the pine wood nematode male worm body diameter of body diameter and other three kinds of media cultivations that dead callus, dead bacterium add the pine wood nematode male worm of callus culture alive with dead bacterium all has notable difference.Above presentation of results with dead bacterium add dead callus, dead bacterium adds callus culture pine wood nematode alive more can promote pine wood nematode male worm body diameter than other three kinds of developing mediums growth.
The different developing mediums of table 14 are to the influence of pine wood nematode male imago volume
| Developing medium | N | Volume averaging value ± standard deviation (mm 3) |
| The dead bacterium of the callus dead bacterium of bacterium alive that lives adds the dead bacterium of dead callus and adds callus alive | 20 20 20 20 20 | 0.00010222＋0.000032505a 0.00012657±0.000032311b 0.00015041±0.000030316c 0.00017493±0.000037205d 0.00018987±0.000038026d |
As shown in Table 14: adding the mean value maximum of volume that callus alive, dead bacterium add the pine wood nematode male worm of dead callus culture with dead bacterium, is with dead bacterium with the mean value minimum of the volume of the pine wood nematode male worm of the pine wood nematode male worm usefulness callus culture alive of the microbial culture of living then successively.From the test of significance result as can be seen: adding the pine wood nematode male worm volume of volume and other three kinds of media cultivations that dead callus, dead bacterium add the pine wood nematode male worm of callus culture alive with dead bacterium all has notable difference; Illustrate with dead bacterium add callus alive, dead bacterium adds dead callus culture pine wood nematode more can promote pine wood nematode male worm volume than other three kinds of media increase.
The result of comprehensively long with the upper body, body diameter and volume is as can be seen: with dead bacterium add that the body that callus alive, dead bacterium add the pine wood nematode male worm of dead callus culture is grown, the mean value of body diameter and volume all is maximum, and the pine wood nematode male worm body of cultivating with other three kinds of media is long, body diameter and volume all have notable difference, illustrate with dead bacterium add dead callus, extremely bacterium adds callus culture pine wood nematode alive and more can promote growing of pine wood nematode male worm than other three kinds of media.
Comprehensive above each experimental result as seen,
From the result of study of black pine callus to the influence of pine wood nematode egg laying amount, no matter be through the black pine callus of freezing treatment inactivation or the black pine callus of living, compare with sterilized water, both can both promote laying eggs of pine wood nematode significantly; But do not have notable difference between the egg laying amount of the pine wood nematode of cultivating with both, illustrate the black pine callus whether inactivation the egg laying amount of pine wood nematode is had no significant effect.
All there were significant differences with the pine wood nematode egg laying amount of cultivating with sterilized water for the egg laying amount of the pine wood nematode of cultivating respectively with the dead Pseudomonas fluorescens GcM5-1A after trichloromethane is handled and the Pseudomonas fluorescens GcM5-1A that lives, illustrates that the both has significant promoter action to the egg laying amount of pine wood nematode; But do not have significant difference between the egg laying amount of the pine wood nematode of cultivating with both respectively, illustrate Pseudomonas fluorescens GcM5-1A whether death the egg laying amount of pine wood nematode is had no significant effect.
From respectively separately with dead black pine callus, separately with dead Pseudomonas fluorescens, dead Pseudomonas fluorescens add dead callus, the callus of living adds Pseudomonas fluorescens alive, dead callus adds Pseudomonas fluorescens alive and the egg laying amount situation of the pine wood nematode of several different medias cultivations such as callus (cultivation has bacterium pine wood nematode ovum) of living as can be seen: compare with sterilized water, more than all developing mediums can both obviously promote laying eggs of pine wood nematode; Wherein add with dead Pseudomonas fluorescens that dead callus, dead bacterium add callus alive, the callus of living adds Pseudomonas fluorescens alive and the egg laying amount of the pine wood nematode that the four kinds of developing mediums of callus (being added with the bacterium ovum) of living are cultivated between do not have notable difference, and four egg laying amounts of cultivating pine wood nematode all want big with the egg laying amount of dead Pseudomonas fluorescens cultivation pine wood nematode separately, but difference is not obvious, but four cultivate the egg laying amount of pine wood nematode all obviously greater than independent pine wood nematode egg laying amount with dead black pine callus culture.Above result shows that in the process of laying eggs of pine worm, the effect of Pseudomonas fluorescens is big than the effect of black pine callus; Add dead black pine callus with dead Pseudomonas fluorescens and may more can promote laying eggs of axenic pine wood nematodes was studied with black pine callus culture axenic pine wood nematodes was studied than independent.
To using the black pine callus of living respectively, dead Pseudomonas fluorescens, Pseudomonas fluorescens alive and dead Pseudomonas fluorescens add dead callus, it is long that dead bacterium adds the female male imago body of pine wood nematode of the first-generation that five kinds of media of callus alive cultivate and the s-generation, body diameter and volume are observed mensuration, the result shows, no matter be the first-generation or the s-generation, add dead callus with dead Pseudomonas fluorescens, it is long that dead bacterium adds the body of the female male imago of pine wood nematode of callus culture alive, body diameter and volume are all obviously long greater than the body of the female male imago of cultivating with other three kinds of media of pine wood nematode, body diameter and volume, three kinds of media of explanation and other are compared, and dead Pseudomonas fluorescens adds dead callus, dead bacterium adds callus alive and can obviously promote growing of pine wood nematode.
From the experiment result of study as can be seen, adding dead black pine callus, dead bacterium with dead Pseudomonas fluorescens and add callus culture axenic pine wood nematodes was studied alive and more can promote the egg laying amount of pine wood nematode and its to grow with the black pine callus culture axenic pine wood nematodes was studied of living more separately, is the method that a kind of ratio is better cultivated axenic pine wood nematodes was studied with work black pine callus separately.This method is than better aspect nematode egg laying amount and growth and breeding with black pine callus culture axenic pine wood nematodes was studied separately.
(4) embodiment:
Below by specific embodiment the present invention is further described
Embodiment 1
1, the cultivation and the processing of Pseudomonas fluorescens (Pseudomonas fluorescens) GcM5-1A bacterial strain: the preparation of (1) NA slant medium: claim 32g nutrient agar medium powder (NA) to be dissolved in the 1000ml distilled water, boil, be sub-packed in the test tube, back at 121 ℃, 1.05kg/cm
2Autoclave in the 15min that sterilizes, contra bevel, standby.(2) actication of culture: Pseudomonas fluorescens (Pseudomonas fluorescens) the GcM5-1A bacterial strain that will in 4 ℃ of refrigerators, preserve, receive on the slant medium under the aseptic condition, be placed in 27 ℃ of constant incubators and cultivate 2d, after put into 4 ℃ of refrigerator and cooled and hide and preserve, standby.(3) cultivation of bacterium: the Pseudomonas fluorescens GcM5-1A bacterial strain that will in 4 ℃ of refrigerators, preserve, in the aseptic processing room, get 1 ring with transfering loop, insert among the NA slant medium 50ml, place 30 ℃ of constant temperature oscillators (128r/min) to go up and cultivate 48h.(4) preparation of dead Pseudomonas fluorescens
A. in sterilisable chamber, soak an amount of trichloromethane (soak into but do not drip) with the absorbent cotton of the bacterium of going out and fill in the test tube mouth, after tampon beyond the Great Wall; Place about 2h in the sterilisable chamber;
B.2h after, be soaked with trichloromethane absorbent cotton, the test tube mouth is opened wide with the aseptic nipper taking-up, in sterilisable chamber, place 2-4h, treat the trichloromethane volatilization to the greatest extent in the test tube after, get 1 ring lawn with transfering loop, insert on the NA slant medium that has prepared, place 28 ℃ constant incubator to cultivate 48h;
C.48h the back observes on the above-mentioned NA slant medium whether bacterial growth is arranged, if do not have, shows that then bacterium is killed by trichloromethane, and dead bacterium can be standby; Otherwise abandoning this experiment also carries out again.
2, cultivate the black pine callus
(1) culture medium preparation
Adopting the 1/2MS substratum is minimum medium, additional KT 4.0mg/L, BA 4.0mg/L, sucrose 30g/L is 5.86 with the HCl of 1mol/L and the NaOH adjusting pH value of 1mol/L, adds agar powder 6.5g/L again, be sub-packed in after the heating for dissolving in the triangular flask of 100mL, in 121 ℃, 1.05kg/cm
2Sterilization 15min obtains the substratum of black pine callus induction.
Adopting the 1/2MS substratum is minimum medium, additional NAA 0.5mg/L, IBA 0.1mg/L, GA
31.0mg/L, 2,4-D 1.0mg/L, LH 100mg/L, sucrose 30g/L is 5.86 with the HCl of 1mol/L and the adjusting pH value of 1mol/LNaOH, adds agar powder 6.5g/L again, be sub-packed in after the heating for dissolving in the triangular flask of 100mL, in 121 ℃, 1.05kg/cm
2Sterilization 15min obtains the substratum that black pine callus succeeding transfer culture is used.If do not add agar powder after regulating the pH value, obtain the liquid nutrient medium that the unicellular shaking culture of black pine is used after the packing sterilization.
(2) the black pine callus induces
The black pine seed is contained in respectively in the small beaker of 50mL, adds a small amount of liquid detergent, seal tight rim of a cup, under flowing water, rinse well with gauze.Shell the back with aseptic water washing 3 times, 75% ethanolic soln surface sterilization 3 times, each 40s uses aseptic water washing 3 times again, afterwards with 0.1% mercuric chloride solution processing 4min, aseptic water washing 5 times.On Bechtop, use the tweezers of the bacterium of going out in the culture dish of bacterium that went out, to divest endosperm, take out mature embryo, it is inserted callus inducing medium, every bottle graft 4-6, place dark culturing in 27 ℃ of incubators.Through about 10d, with well-grown, individuality promptly not contaminated and that brown stain do not take place is transferred to and is kept in the subculture medium and breed.
(3) succeeding transfer culture of black pine callus
In the aseptic processing room, callus is cut into the tissue block of soya bean size, insert on the subculture medium, insert the 3-4 piece in each triangular flask, place 27 ℃ of thermostat containers secretly to cultivate, once every the 20-30d succeeding transfer culture.
3. pine wood nematode is as good as cultivation
(1) preparation of axenic pine wood nematodes was studied ovum
A. separate pine wood nematode: will go up the pine wood nematode of cultivating Botrytis cinerea (Botryis cinerea) and separate with the graceful funnel method of shellfish.Centrifugal the pine wood nematode that is separated to, identify, standby;
B. with above-mentioned centrifugal, the pine wood nematode after the evaluation is added on the 2% good dull and stereotyped nutrient agar of prepared beforehand, seals culture dish with sealing film, inserts in 27 ℃ the constant incubator and cultivates 6-24h;
C. in the training period, observe the situation of laying eggs of pine wood nematode; When the nematode egg laying amount reaches the experiment necessary requirement, under aseptic condition, inhale an amount of sterilized water with the suction pipe of the bacterium of going out pine wood nematode on the nutrient agar and ovum are wherein all washed in the centrifuge tube of the bacterium of going out, splash into 30% H
2O
2(with the volume ratio of ovum suspension be 1: 1), under 15 ℃, leave standstill 10min, examine under a microscope the death condition of nematode;
D. treat that nematode is most of dead, with aseptic water washing 3-5 time, add sterilized water at every turn after, be placed on centrifugal 3-4min on the whizzer after shaking up, wait to wash down in the centrifuge tube behind the hydrogen peroxide, filter with 200 aseptic order stainless steel filter sieve, repeat to filter 4-6 time, till most of nematode is filtered out;
E. check that with the NA slant medium for preparing in advance line eggs has or not bacterium; If bacterium, then abandon this experiment and carry out again.
(2) preparation nutrient solution
F. under the aseptic condition, take by weighing the above-mentioned dead bacterium of above-mentioned callus of 10mg and 0.1mg, add the 2ml sterilized water, or prepare nutrient solution according to the above ratio.
(3) cultivation of pine wood nematode
G. in above-mentioned nutrient solution, add the axenic pine wood nematodes was studied ovum that has prepared with aseptic straw
H. above-mentioned nutrient solution is placed under 40 ℃, secretly cultivate 20d after, required axenic pine wood nematodes was studied.
Embodiment 2
1, the cultivation and the processing of Pseudomonas fluorescens (Pseudomonas fluorescens) GcM5-1A bacterial strain: the preparation of (1) NB liquid nutrient medium: take by weighing 96g nutrient broth (NB) and be dissolved in the 3000ml distilled water, boil, be sub-packed in the triangular flask; At 121 ℃, the 15min that sterilizes in the Autoclave of 1.05kg/cm2, standby.(2) actication of culture.Method is with embodiment 1.(3) cultivation of bacterium: under aseptic condition, the false unit cell GcM5-1A of activatory fluorescence bacterial strain is inserted in the NB liquid nutrient medium,, place 30 ℃ of constant temperature oscillators (128r/min) to go up and cultivate 4d by inserting 1ml activation bacterial strain in every 50ml NB liquid nutrient medium.(4) acquisition of dead bacterium: with the bacterium that step (3) obtains, under aseptic condition, place-40 ℃ of following 12h frozen dryings, take out under the rearmounted room temperature and thaw, carry out lyophilize, get the dead bacterium of aseptic exsiccant.Standby
2, cultivate the black pine callus
Cultivate the black pine callus with the method identical with embodiment 1, then,
With the black pine callus that above step obtains, under aseptic condition, place-40 ℃ of following 12h frozen dryings, take out under the rearmounted room temperature and thaw, carry out lyophilize, aseptic drying dead callus.Standby
3. pine wood nematode is as good as cultivation
(1) preparation of axenic pine wood nematodes was studied ovum, method is with embodiment 1.(2) preparation nutrient solution: under the aseptic condition, take by weighing above-mentioned dead callus of 10mg and the above-mentioned dead bacterium of 0.1mg, add the 2ml sterilized water, or prepare nutrient solution according to the above ratio.(3) cultivation of pine wood nematode: in above-mentioned nutrient solution, add the axenic pine wood nematodes was studied ovum that has prepared with aseptic straw.Above-mentioned nutrient solution is placed under 15 ℃, secretly cultivate 20d after, required axenic pine wood nematodes was studied.
Embodiment 3
1, the cultivation of Pseudomonas fluorescens GcM5-1A bacterial strain and processing, method is with embodiment 1
2, cultivate the black pine callus, method is with embodiment 1
Pine wood nematode be as good as cultivation: the preparation of (1) axenic pine wood nematodes was studied ovum, method is with embodiment 1.(2) preparation nutrient solution: under the aseptic condition, take by weighing above-mentioned callus alive of 200mg and the above-mentioned dead bacterium of 0.1mg, add the 2ml sterilized water, or prepare nutrient solution according to the above ratio.(3) cultivation of pine wood nematode: in above-mentioned nutrient solution, add the axenic pine wood nematodes was studied ovum that has prepared with aseptic straw.Above-mentioned nutrient solution is placed under 23 ℃, secretly cultivate 10d after, required axenic pine wood nematodes was studied.
Embodiment 4
1, the cultivation of Pseudomonas fluorescens GcM5-1A bacterial strain and processing, method is with embodiment 1
2, cultivate the black pine callus, method is with embodiment 1
Pine wood nematode be as good as cultivation: the preparation of (1) axenic pine wood nematodes was studied ovum, method is with embodiment 1.(2) preparation nutrient solution: under the aseptic condition, take by weighing above-mentioned callus alive of 40mg and the above-mentioned dead bacterium of 0.1mg, add the 2ml sterilized water, or prepare nutrient solution according to the above ratio.(3) cultivation of pine wood nematode: in above-mentioned nutrient solution, add the axenic pine wood nematodes was studied ovum that has prepared with aseptic straw.Above-mentioned nutrient solution is placed under 31 ℃, secretly cultivate 10d after, required axenic pine wood nematodes was studied.
Embodiment 5
1, the cultivation of Pseudomonas fluorescens GcM5-1A bacterial strain and processing, method is with embodiment 2
2, cultivate the black pine callus, method is with embodiment 1
Pine wood nematode be as good as cultivation: the preparation of (1) axenic pine wood nematodes was studied ovum, method is with embodiment 1.(2) preparation nutrient solution: under the aseptic condition, take by weighing above-mentioned callus alive of 150mg and the above-mentioned dead bacterium of 0.1mg, add the 2ml sterilized water, or prepare nutrient solution according to the above ratio.(3) cultivation of pine wood nematode: in above-mentioned nutrient solution, add the axenic pine wood nematodes was studied ovum that has prepared with aseptic straw.Above-mentioned nutrient solution is placed under 25 ℃, secretly cultivate 4d after, required axenic pine wood nematodes was studied.
Embodiment 6
1, the cultivation of Pseudomonas fluorescens GcM5-1A bacterial strain and processing, method is with embodiment 2
2, cultivate the black pine callus, method is with embodiment 1
Pine wood nematode be as good as cultivation: the preparation of (1) axenic pine wood nematodes was studied ovum, method is with embodiment 1.(2) preparation nutrient solution: under the aseptic condition, take by weighing above-mentioned callus alive of 80mg and the above-mentioned dead bacterium of 0.1mg, add the 2ml sterilized water, or prepare nutrient solution according to the above ratio.(3) cultivation of pine wood nematode: in above-mentioned nutrient solution, add the axenic pine wood nematodes was studied ovum that has prepared with aseptic straw.Above-mentioned nutrient solution is placed under 28 ℃, secretly cultivate 4d after, required axenic pine wood nematodes was studied.
Embodiment 7
1, the cultivation of Pseudomonas fluorescens GcM5-1A bacterial strain and processing, method is with embodiment 2
2, cultivate the black pine callus, method is with embodiment 1
Pine wood nematode be as good as cultivation: the preparation of (1) axenic pine wood nematodes was studied ovum, method is with embodiment 1.(2) preparation nutrient solution: under the aseptic condition, take by weighing above-mentioned callus alive of 120mg and the above-mentioned dead bacterium of 0.1mg, add the 2ml sterilized water, or prepare nutrient solution according to the above ratio.(3) cultivation of pine wood nematode: in above-mentioned nutrient solution, add the axenic pine wood nematodes was studied ovum that has prepared with aseptic straw.Above-mentioned nutrient solution is placed under 15 ℃, secretly cultivate 20d after, required axenic pine wood nematodes was studied.
Embodiment 8
1, the cultivation of Pseudomonas fluorescens GcM5-1A bacterial strain and processing, method is with embodiment 1
2, cultivate and obtain dead black pine callus, method is with embodiment 2
Pine wood nematode be as good as cultivation: the preparation of (1) axenic pine wood nematodes was studied ovum, method prepares nutrient solution with embodiment 1 (2): under the aseptic condition, take by weighing above-mentioned dead callus of 0.5mg and the above-mentioned dead bacterium of 0.1mg, add the 2ml sterilized water, or prepare nutrient solution according to the above ratio.(3) cultivation of pine wood nematode adds the axenic pine wood nematodes was studied ovum that has prepared with aseptic straw in above-mentioned nutrient solution.Above-mentioned nutrient solution is placed under 40 ℃, secretly cultivate 20d after, required axenic pine wood nematodes was studied.
Embodiment 9
1, the cultivation of Pseudomonas fluorescens GcM5-1A bacterial strain and processing, method is with embodiment 2
2, cultivate and obtain dead black pine callus, method is with embodiment 1, obtain the black pine callus after, lyophilize, aseptic exsiccant callus.
Pine wood nematode be as good as cultivation: the preparation of (1) axenic pine wood nematodes was studied ovum, method is with embodiment 1.(2) preparation nutrient solution: under the aseptic condition, take by weighing the above-mentioned dead Pseudomonas fluorescens of dead callus of the above-mentioned exsiccant of 2mg and 0.1mg, add the 2ml sterilized water, or prepare nutrient solution according to the above ratio.(3) cultivation of pine wood nematode: in above-mentioned nutrient solution, add the axenic pine wood nematodes was studied ovum that has prepared with aseptic straw.Above-mentioned nutrient solution is placed under 23 ℃, secretly cultivate 10d after, required axenic pine wood nematodes was studied.
Embodiment 10
1, the cultivation of Pseudomonas fluorescens GcM5-1A bacterial strain and processing, method is with embodiment 1
2, cultivate and obtain dead black pine callus, method is with embodiment 2.After obtaining the black pine callus, lyophilize gets the dead callus of aseptic exsiccant.
Pine wood nematode be as good as cultivation: the preparation of (1) axenic pine wood nematodes was studied ovum, method is with embodiment 1.(2) preparation nutrient solution: under the aseptic condition, take by weighing the above-mentioned dead bacterium of dead callus of the above-mentioned exsiccant of 7.5mg and 0.1mg, add the 2ml sterilized water, or prepare nutrient solution according to the above ratio.(3) cultivation of pine wood nematode: in above-mentioned nutrient solution, add the axenic pine wood nematodes was studied ovum that has prepared with aseptic straw.Above-mentioned nutrient solution is placed under 31 ℃, secretly cultivate 10d after, required axenic pine wood nematodes was studied.
Embodiment 11:
1, the cultivation of Pseudomonas fluorescens GcM5-1A bacterial strain and processing, method is with embodiment 1.
2, cultivate and obtain dead black pine callus, method is with embodiment 2.After obtaining the black pine callus, lyophilize gets aseptic exsiccant callus.
Pine wood nematode be as good as cultivation: the preparation of axenic pine wood nematodes was studied ovum, method is with embodiment 1.(2) preparation nutrient solution: under the aseptic condition, take by weighing the above-mentioned dead bacterium of dead callus of the above-mentioned exsiccant of 4mg and 0.1mg, add the 2ml sterilized water, or prepare nutrient solution according to the above ratio.(3) cultivation of pine wood nematode: in above-mentioned nutrient solution, add the axenic pine wood nematodes was studied ovum that has prepared with aseptic straw.Above-mentioned nutrient solution is placed under 25 ℃, secretly cultivate 4d after, required axenic pine wood nematodes was studied.
Embodiment 12
1, the cultivation of Pseudomonas fluorescens GcM5-1A bacterial strain and processing, method is with embodiment 1.
2, cultivate and obtain dead black pine callus, method is with embodiment 2.After obtaining the black pine callus, lyophilize gets aseptic exsiccant callus.
Pine wood nematode be as good as cultivation: the preparation of (1) axenic pine wood nematodes was studied ovum, method is with embodiment 2.(2) preparation nutrient solution: under the aseptic condition, take by weighing the above-mentioned dead bacterium of dead callus of the above-mentioned exsiccant of 6mg and 0.1mg, add the 2ml sterilized water, or prepare nutrient solution according to the above ratio.(3) cultivation of pine wood nematode: in above-mentioned nutrient solution, add the axenic pine wood nematodes was studied ovum that has prepared with aseptic straw.Above-mentioned nutrient solution is placed under 28 ℃, secretly cultivate 4d after, required axenic pine wood nematodes was studied.
Embodiment 13
1, the cultivation of Pseudomonas fluorescens GcM5-1A bacterial strain and processing, method is with embodiment 1
2, cultivate and obtain dead black pine callus: after obtaining the black pine callus with embodiment 1 step, with fresh aseptic callus under aseptic condition, by monobromethane the black pine callus that obtains in the step 4 is handled (method is with embodiment 1), get aseptic dead callus.Drying gets the dead callus of aseptic exsiccant, and is standby.
Pine wood nematode be as good as cultivation: the preparation of (1) axenic pine wood nematodes was studied ovum, adopt known method to cultivate pine wood nematode, treat that it lays eggs, adopt mercuric chloride to separate and obtain aseptic ovum.Its concrete grammar is: line eggs is soaked 30s in 75% ethanol, move into 0.1%HgCI
2Solution surface sterilization 1min, aqua sterilisa is changed clothes 3 times. get the axenic pine wood nematodes was studied ovum.(2) preparation nutrient solution: under the aseptic condition, take by weighing the above-mentioned dead bacterium of dead callus of the above-mentioned exsiccant of 10mg and 0.01mg, add the 2ml sterilized water, or prepare nutrient solution according to the above ratio.(3) cultivation of pine wood nematode: in above-mentioned nutrient solution, add the axenic pine wood nematodes was studied ovum that has prepared with aseptic straw, above-mentioned nutrient solution placed under 26 ℃, secretly cultivate 5d after, required axenic pine wood nematodes was studied.
Certainly, under inventive concept of the present invention, the present invention has multiple form of implementation, reads as, those skilled in the art and need not pay creative work behind this specification sheets and can reveal again, has not just described in detail at this.
Also be pointed out that at this, continue to use inventive concept of the present invention, the bacterium alive that callus alive that obtains with step 4 in the technology of the present invention solution or the dead callus that step 5 was obtained and step 1 are obtained by with step 6 in the germ-carrying pine wood nematode of same ratio prepared culture, for the germ-carrying pine wood nematode technology of existing cultivation, more effective equally, its individual size, breeding potential, output and quality are all higher than prior art.
Claims (12)
1, a kind of cultural method of axenic pine wood nematodes was studied comprises following step:
(1) cultivate Pseudomonas fluorescens (Pseudomonasfluorescens) GcM5-1A CCTCC No:M 204065 with conventional culturing bacterium method and obtain the bacteria in viable somatic cells, thereby and it carried out inactivation treatment obtain dead cell, standby;
(2) preparation axenic pine wood nematodes was studied ovum is standby;
(3) cultivate the black pine callus, standby;
(4) under the aseptic condition, be mixed with different nutrient solutions with each lay-by material mixing of step (1), (3) acquisition and in following ratio and sterilized water:
The black pine callus: dead bacterium mass ratio is 100 ~ 2000: 1
(5) in the nutrient solution that step (4) obtains, add the axenic pine wood nematodes was studied ovum for preparing in the step (2), place temperature: 15-40 ℃, secretly cultivate 4-20d after, required axenic pine wood nematodes was studied.
2, a kind of cultural method of axenic pine wood nematodes was studied comprises following step:
(1) cultivate Pseudomonas fluorescens (Pseudomonas fluorescens) GcM5-1A CCTCC No:M 204065 with conventional culturing bacterium method and obtain the bacteria in viable somatic cells, thereby and it carried out inactivation treatment obtain dead cell, standby;
(2) preparation axenic pine wood nematodes was studied ovum is standby;
(3) cultivate the black pine callus;
(4) thus above-mentioned acquisition black pine callus is carried out inactivation treatment obtains the dead black pine callus of aseptic exsiccant, standby;
(5) under the aseptic condition, each lay-by material that obtains with step (1), (4) is mixed with different nutrient solutions by following mixed and with sterilized water:
Dead callus: dead bacterium mass ratio is 5 ~ 100: 1
(6) in the nutrient solution that step (5) obtains, add the axenic pine wood nematodes was studied ovum for preparing in the step (2), place temperature: 15-40 ℃, secretly cultivate 4-20d after, required axenic pine wood nematodes was studied.
3, according to the cultural method of claim 1 or 2 arbitrary described axenic pine wood nematodes was studieds, it is characterized in that: described step (1) thus in adopt trichloromethane method in the chemical process that the bacteria in viable somatic cells that is obtained is carried out inactivation treatment to obtain dead cell, specific practice is as follows:
A. the somatic cells that cultivation is obtained places test tube or flask, soaks an amount of trichloromethane with the absorbent cotton of bacterium of going out, and soaks into but does not drip, and fills in test tube or flask mouth, after tampon beyond the Great Wall; Place about 2-4h in the sterilisable chamber;
B. take out with aseptic nipper and be soaked with trichloromethane absorbent cotton, test tube mouth or flask mouth are opened wide, in sterilisable chamber, place 2-4h, after treating the trichloromethane volatilization to the greatest extent in the test tube, get 1 ring lawn with transfering loop, insert on the NA slant medium that has prepared, place 28 ℃ constant incubator to cultivate 48h;
C.48h the back observes on the above-mentioned NA slant medium whether bacterial growth is arranged, if do not have, shows that then bacterium is killed by trichloromethane, and dead bacterium can be standby; Otherwise sterilization again.
4, according to the cultural method of claim 1 or 2 arbitrary described axenic pine wood nematodes was studieds, it is characterized in that: adopt H in the step (2)
2O
2Obtain worm's ovum thereby kill nematode and separate, specific practice is as follows:
A. separate pine wood nematode: will go up the pine wood nematode of cultivating Botrytis cinerea (Botryis cinerea) and separate, centrifugal the pine wood nematode that is separated to, identify, standby;
B. with above-mentioned centrifugal, the pine wood nematode after the evaluation is added on the 2% good dull and stereotyped nutrient agar of prepared beforehand, seals culture dish with sealing film, inserts in 27 ℃ the constant incubator and cultivates 6-24h;
C. in the training period, observe the situation of laying eggs of pine wood nematode; When the nematode egg laying amount reaches the experiment necessary requirement, under aseptic condition, inhale an amount of sterilized water with the suction pipe of the bacterium of going out pine wood nematode on the nutrient agar and wherein ovum are all washed in the centrifuge tube of the bacterium of going out, splash into volume ratio with the ovum suspension and be 1: 1 30% H
2O
2, under 15 ℃, leave standstill 10min, examine under a microscope the death condition of nematode;
D. treat that nematode is most of dead, with aseptic water washing 3-5 time, add sterilized water at every turn after, be placed on centrifugal 3-4min on the whizzer after shaking up, wait to wash down in the centrifuge tube behind the hydrogen peroxide, filter with 200 aseptic order stainless steel filter sieve, repeat to filter 4-6 time, till most of nematode is filtered out;
E. check that with the NA slant medium for preparing in advance line eggs has or not bacterium; If bacterium, then abandon this experiment and carry out again.
5, the cultural method of axenic pine wood nematodes was studied according to claim 1 is characterized in that: in the step (5), described culture temperature is 23-31 ℃, and dark incubation time is 4-10d.
6, the cultural method of axenic pine wood nematodes was studied according to claim 2 is characterized in that: in the step (6), described culture temperature is 23-31 ℃, and dark incubation time is 4-10d.
7, the cultural method of axenic pine wood nematodes was studied according to claim 1 is characterized in that: in the step (5), described culture temperature is 25-28 ℃, and dark incubation time is 4-5d.
8, the cultural method of axenic pine wood nematodes was studied according to claim 2 is characterized in that: in the step (6), described culture temperature is 25-28 ℃, and dark incubation time is 4-5d.
9, the cultural method of axenic pine wood nematodes was studied according to claim 1 is characterized in that: in the preparation process of described nutrient solution, callus is 400～1500: 1 with the mass ratio of dead bacterium.
10, the cultural method of axenic pine wood nematodes was studied according to claim 1 is characterized in that: in the preparation process of described nutrient solution, callus is 800～1200: 1 with the mass ratio of dead bacterium.
11, the cultural method of axenic pine wood nematodes was studied according to claim 2 is characterized in that: in the preparation process of described nutrient solution, the dead callus of aseptic exsiccant is 20～75: 1 with the mass ratio of dead bacterium.
12, the cultural method of axenic pine wood nematodes was studied according to claim 2 is characterized in that: in the preparation process of described nutrient solution, the dead callus of aseptic exsiccant is 40～60: 1 with the mass ratio of dead bacterium.
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| CN101334363B (en) * | 2008-01-09 | 2010-04-14 | 青岛大学 | Method for detecting pine wood nematode based on pseudomonas fluorescent flagellum protein |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102084849B (en) * | 2010-11-19 | 2012-06-13 | 南京林业大学 | Method for preparing bacteria-free pine wood nematodes |
| CN102433295B (en) * | 2011-08-24 | 2014-07-30 | 中国科学院东北地理与农业生态研究所 | Living nematoda egg purification and extraction agent as well as its application method |
| CN102613406A (en) * | 2012-03-28 | 2012-08-01 | 浙江农林大学 | Preparation method of artificial feeding liquid for pine wood nematode |
| CN102578433A (en) * | 2012-03-28 | 2012-07-18 | 浙江农林大学 | Formula of pine wood nematode feed and preparation method |
| CN103004592B (en) * | 2012-12-10 | 2013-12-25 | 华南农业大学 | Method for breeding, culturing and preserving paratylenchus by using carrot callus |
| CN109169534A (en) * | 2018-10-15 | 2019-01-11 | 南开大学 | Nematode solid medium and its preparation method and application |
| CN109601482B (en) * | 2018-12-07 | 2021-06-25 | 江苏科技大学 | Method for preparing sterile pine wood nematodes |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101334363B (en) * | 2008-01-09 | 2010-04-14 | 青岛大学 | Method for detecting pine wood nematode based on pseudomonas fluorescent flagellum protein |
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